Selectively promiscuous opioid ligands: Discovery of high affinity/low efficacy opioid ligands with substantial nociceptin opioid peptide receptor affinity

Article abstract of DOI:10.1021/jm401964y

Emerging clinical and preclinical evidence suggests that a compound displaying high affinity for μ, κ, and δ opioid (MOP, KOP, and DOP) receptors and antagonist activity at each, coupled with moderate affinity and efficacy at nociceptin opioid peptide (NOP) receptors will have utility as a relapse prevention agent for multiple types of drug abuse. Members of the orvinol family of opioid ligands have the desired affinity profile but have typically displayed substantial efficacy at MOP and or KOP receptors. In this study it is shown that a phenyl ring analogue (1d) of buprenorphine displays the desired profile in vitro with high, nonselective affinity for the MOP, KOP, and DOP receptors coupled with moderate affinity for NOP receptors. In vivo, 1d lacked any opioid agonist activity and was an antagonist of both the MOP receptor agonist morphine and the KOP receptor agonist ethylketocyclazocine, confirming the desired opioid receptor profile in vivo.

Full text of DOI:10.1021/jm401964y

Article
pubs.acs.org/jmc
Selectively Promiscuous Opioid Ligands: Discovery of High Affinity/
Low Efficacy Opioid Ligands with Substantial Nociceptin Opioid
Peptide Receptor Affinity
Vinod Kumar,†,⊥ Irna E. Ridzwan,† Konstantinos Grivas,§ John W. Lewis,† Mary J. Clark,‡
Claire Meurice,‡ Corina Jimenez-Gomez,‡ Irina Pogozheva,∥ Henry Mosberg,∥ John R. Traynor,‡
and Stephen M. Husbands*,†
†Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY, U.K.
‡Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, United States
§School of Chemistry, University of Bristol, Bristol, BS8 1TS, U.K.
∥College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States
S
* Supporting Information
ABSTRACT: Emerging clinical and preclinical evidence
suggests that a compound displaying high affinity for μ, κ,
and δ opioid (MOP, KOP, and DOP) receptors and
antagonist activity at each, coupled with moderate affinity
and efficacy at nociceptin opioid peptide (NOP) receptors will
have utility as a relapse prevention agent for multiple types of
drug abuse. Members of the orvinol family of opioid ligands
have the desired affinity profile but have typically displayed
substantial efficacy at MOP and or KOP receptors. In this
study it is shown that a phenyl ring analogue (1d) of
buprenorphine displays the desired profile in vitro with high,
nonselective affinity for the MOP, KOP, and DOP receptors coupled with moderate affinity for NOP receptors. In vivo, 1d
lacked any opioid agonist activity and was an antagonist of both the MOP receptor agonist morphine and the KOP receptor
agonist ethylketocyclazocine, confirming the desired opioid receptor profile in vivo.
Buprenorphine’s KOP and DOP receptor antagonism and
NOP receptor partial agonism appear to contribute to its
demonstrated potential as a treatment for cocaine and ethanol
abuse and dependence in addition to its approved use in opiate
abuse and dependence that is derived from its MOP receptor
partial agonism.8
Use of buprenorphine to treat cocaine and alcohol abuse
would not be allowed in patients without concurrent opiate
abuse problems, since buprenorphine treatment supports a
significant level of MOP receptor dependence.7 Thus, one of
our medicinal chemistry objectives has been to discover, among
structural analogues of buprenorphine, ligands having MOP
and KOP receptor antagonist and NOP receptor agonist or
partial agonist activity. Such a compound should be more
widely useful than buprenorphine in the treatment of cocaine
and alcohol abuse and dependence.
INTRODUCTION
■
The orvinols are a group of ring-C bridged epoxymorphinan
compounds that were originally synthesized by Bentley and co-
workers1−3 and developed by Reckitt and Colman.4 The most
studied members of the series include the very potent opiate
antagonist diprenorphine (1a)1 and buprenorphine (1b)
(Chart 1), the clinical analgesic and treatment agent for opiate
abuse and addiction.5−8
Buprenorphine displays a unique and complex pharmacology
derived from the manner in which it binds to opioid
receptors.9−11 At the μ opioid (MOP) receptor it is a partial
agonist with high affinity and slow onset and offset, thus having
“irreversible” characteristics, manifested in its long duration of
action and the mildness of abstinence effects when the drug is
withdrawn following chronic administration. At the other
opioid receptors, κ (KOP) and δ (DOP) buprenorphine has
negligible efficacy and is a potent antagonist of KOP and DOP
receptor agonists. In addition to its binding to these classical
opioid receptors, buprenorphine also binds as a partial agonist
of moderate affinity to the nociceptin opioid peptide (NOP)
receptor that, though having a high degree of amino acid
sequence homology with the classical opioid receptors,
nevertheless has negligible affinity for most opioid ligands.
Structure−activity relationship studies in orvinols of
structures 1 and 2 (Chart 1) based solely on in vivo
antinociception data demonstrated that in structure 1 only
when R1 and R2 were H or methyl was MOPr antagonist
Received: December 20, 2013
Published: April 24, 2014
© 2014 American Chemical Society
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Chart 1
activity shown without accompanying antinociceptive activ-
ity.1,12 These studies did not rule out the possibility that these
MOPr antagonists may have had low efficacy KOPr and DOPr
activity below the level required to produce an antinociceptive
response in the mouse antiwrithing test. Nevertheless it was
clear that when R1 and R2 in structure 1 were alkyl groups
larger than methyl, substantial MOPr activity was normally
found. Further insight into orvinol SAR has recently been
provided with the first publication of affinity and efficacy data
for a sizable series of predominantly branched-chain orvinols.13
Substantial efficacy for MOPr and KOPr was found to be the
norm. KOPr agonist efficacy, which would translate in humans
as debilitating dysphoric side effects, is dominant. Twenty-five
of the 39 compounds tested for KOPr efficacy showed full
KOPr agonism (>85% of the response to the full KOPr agonist,
U69593), and the others showed a partial KOPr agonist
response (30−75% of U69593). The notable exception was
buprenorphine, which gave zero KOPr agonist response. Three
out of the 40 compounds tested had zero MOPr efficacy, and
three had 80% or greater (compared to the full MOPr agonist
DAMGO = 100%). The majority, including buprenorphine,
demonstrated efficacy between these limits. A CoMFA
(comparative molecular field analysis) model developed using
this series of ligands suggested that a bulky group immediately
adjacent to C20 was key to obtaining low efficacy at KOP
receptors when R1 in 1 was larger than methyl.
evaluate a number of analogues. The results of these studies are
reported below along with C20-ethyl homologues, as the effect
of increasing lipophilicity/bulk through manipulation of the
C20-methyl group has also not been explored previously.
SYNTHESIS
■
All tertiary alcohols were accessed by Grignard addition to the
known methyl ketone (8), itself prepared by the recently
reported methods of Greedy et al. (Scheme 1).13 No addition
could be achieved with pyridyl Grignard reagents, and so
pyridyl lithium addition to the ketone was attempted. This was
successful but interestingly gave the opposite diastereomer to
that expected (Scheme 1), i.e., opposite to that obtained from
standard Grignard addition (confirmed by X-ray crystallog-
raphy; see Supporting Information). It appears that the reactive
aryllithium addition follows the Felkin−Ahn model14 such that
the carbonyl oxygen would be oriented between the large (C6)
and medium (C8) neighboring groups, orthogonal to the large
(C6) group. Attack of the nucleophile then occurs anti to C6
(Figure 1a). Addition of the less reactive Grignard reagents
appears to require initial formation of a six-membered chelate
ring (Figure 1b), providing an ordered and activated complex,
with nucleophilic addition then occurring from the less
hindered (C7-H) face to afford the other epimer. Access to
the secondary phenyl alcohols (1e, 1g, 2a, 2b) was via the
known aldehydes (11a,b)13 (Scheme 2). Addition of phenyl-
magnesium bromide to 11a and 11b gave 12a and 12b,
respectively, the opposite diastereoisomer to that obtained on
Grignard addition to the methyl ketone (as shown in Scheme
1). Presumably, addition to the more reactive aldehyde does
not require formation of the active complex, and so addition
This led to an investigation of orvinol structures having a
large lipophilic moiety directly attached to C20, during which
we synthesized a set of phenyl orvinols (1d−f, 2a−c). One of
these ligands (1d) was proven to lack any significant MOPr or
KOPr efficacy in vitro or in vivo. This led us to synthesize and
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a
As expected, in these assays the new compounds all had high
affinity for all MOP, DOP, and KOP receptors with no
evidence of any selectivity for an individual receptor type
(Table 1). Changing R2 from methyl to ethyl (1d to 1f) had no
effect on binding affinity, whereas secondary alcohol C20
groups were associated with a slightly lower affinity than
tertiary alcohol groups (e.g., 1e versus 1d, 1f), particularly at
KOP and DOP receptors. The diasereoisomers 2a and 2b both
displayed nonselective binding, though absolute affinities could
not be compared because 2b was evaluated in a separate assay.
Nevertheless all binding affinities were in the nanomolar range.
In the [35S]GTPγS assay for functional opioid activity at
MOP, DOP, and KOP receptors using methods reported
previously,15 only 1d was a potent MOP and KOP receptor
antagonist (Table 1); it failed to show MOP receptor agonist
activity and had very low level KOP receptor efficacy (Table 2).
The other phenyl orvinols were MOP receptor partial agonists
of efficacy ranging from the low (1f, 2a) to moderately high
(2c). They were generally high efficacy KOP receptor partial to
full agonists. They showed a similar pattern of agonist efficacy
at DOP receptors, at which 1d was also a partial agonist of
modest efficacy. It was striking that 1f and 2c which differ
structurally only in the 6,14-bridge differed markedly in efficacy
for MOP, DOP, and KOP receptor types with the etheno-
bridged ligand 2c having markedly higher efficacy.
Scheme 1
a
Reagents and conditions: (i) RMgBr, THF, rt; (ii) 2-pyridyllithium or
4-pyridyllithium, Et2O, THF, −78 °C → rt; (iii) PrSNa, HMPA, 110
°C or L-selectride, THF, reflux.
follows the Felkin−Ahn model. Subsequent oxidation to 13a
and 13b followed by then reduction with lithium aluminum
hydride provided the opposite diastereoisomers (14a and 14b).
Finally, 3-O-demethylation gave the desired phenolic products
1e and 2a.
The effect of replacing the R2 methyl group in
buprenorphine (1b) by ethyl was also investigated. The
homologue (1c) had comparable affinities at MOP, DOP,
and KOP receptors. Affinity for NOP receptors was also
measured and compared with that of buprenorphine; again
affinities were comparable (Table 1). The etheno analogue
(2d) of 1c was also evaluated. Binding affinity for MOP, DOP,
and KOP receptors was similar to that of 1c, but NOP receptor
affinity was lower. In [35S]GTPγS assays 1c had somewhat
higher MOP receptor efficacy than buprenorphine while the
etheno analogue (2d) was an almost full MOP receptor agonist
though with very modest potency. 1c had high KOP receptor
RESULTS
■
In Vitro. Opioid receptor binding affinities of the first series
of phenyl orvinol analogues (1d−g, 2a−d) were determined by
displacement of [3H]DAMGO, [3H]-DPDPE, [3H]U69,593,
and [3H]N/OFQ from human opioid receptors transfected into
Chinese hamster ovary (CHO) cells. Details of these assays
have been described previously.15
Figure 1. Nucleophilic addition (a) without chelation and (b) with chelation control.
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a
Scheme 2
a
Reagents and conditions: (i) PhMgBr, THF, rt; (ii) (COCl)2, DMSO, DMSO, CH2Cl2, −78 °C; (iii) LiAlH4, ether; (iv) PrSNa, HMPA, 110 °C or
L-selectride, THF, reflux.
Table 1. Binding Affinities (Ki, nM) and stimulation of [35S]GTPγS Binding of Series 1 and 2 to Opioid Receptors
ae
,
b
ce
,
Ki, nM
EC50, nM; % stim or [Ke, nM]
MOP
KOP
DOP
NOP
MOP
KOP
DOP
1b
1c
1d
1e
1f
1.5 0.80
4.0 0.57
0.71 0.17
1.3 0.39
1.0 0.15
0.82 0.30
4.0 0.63
0.80 0.50
3.2 0.38
4.2 0.60
2.5 1.2
6.1 0.40
0.86 0.02
1.9 0.33
2.6 0.22
0.80 0.05
1.3 0.36
3.2 0.48
0.40 0.0
1.2 0.14
1.2 0.16
77 16
105 4.0
NT
10.2 2.2; 29 1.1
50.2 6.6; 40 3.7
[0.47 0.03]
NS
NS
>10000
0.56 0.01
0.49 0.08
4.4 1.6
183 9.4; 73 17
[0.27 0.03]
2.66 0.34; 34 8.0
4.54 0.58; 90 5.1
8.90 1.8; 30 7.5
0.64 0.17; 113 3.8
1.76 0.46; 55 0.82
NT
NT
10.4 2.7; 32 5.9
18.4 5.7; 18 1.0
1.55 0.28; 37 1.1
2.75 1.05; 18 0.9
NT
1.09 0.0; 67 1.4
249 120; 22 4.4
0.36 0.03; 79 5.0
2.10 0.49; 70 4.6
NT
0.36 0.04
0.88 0.03
3.8 0.74
1.5 0.95
0.95 0.26
0.75 0.11
396 41
NT
1g
2a
2b
2c
2d
NT
d
NT
197 0.21
187 27
31.8 18.5; 56 13
238 19; 84 3.5
56.6 11; 128 2.4
122 63; 57 5.6
10.2 2.4; 123 22
314 11; 115 5.2
a
Displacement of [3H]DAMGO, [3H]-DPDPE, [3H]U69,593, and [3H]N/OFQ from human opioid receptors transfected into Chinese hamster
b
c
ovary (CHO) cells. % maximal stimulation with respect to the standard agonists DAMGO (MOP), U69,593 (KOP), and DPDPE (DOP). Values
in brackets are antagonist Ke values versus the standard agonists DAMGO (MOP), U69,593 (KOP), and DPDPE (DOP). Values are the average
d
SEM from three separate experiments. Binding to Hartley guinea pig brain membranes, Ki (nM) versus [3H]DAMGO, [3H]DPDPE, [3H]U69,593.
e
NS: no stimulation. NT: not tested.
efficacy, whereas buprenorphine is a KOP receptor antagonist;
1c like buprenorphine has no efficacy for DOP receptors. In
that respect there was a remarkable difference between 1c and
the etheno analogue (2d) which was a full DOP receptor
agonist though of low potency.
We followed up on these findings by focusing on close
analogues of 1d to identify which had the desired profile of
MOP and KOP receptor antagonism. To expedite this process,
a primary assay was established whereby compounds were
evaluated in the [35S]GTPγS assay for MOP, KOP, and NOP
receptor efficacy at a very high concentration (10 μM) to
determine peak efficacy at each receptor (Table 2). Nine
phenyl substituted analogues of 1d and six analogues with the
phenyl group of 1d replaced by heteroaryl rings were evaluated.
A limited number of compounds were also evaluated for affinity
at MOP, DOP, and KOP receptors by measuring displacement
of [3H]diprenorphine binding from C6-rat glioma cells
expressing recombinant rat MOP and DOP receptors and
CHO cells expressing recombinant human KOP receptors,
essentially to confirm the expected high affinity of the series at
these receptors. NOP receptor binding affinity was measured
by displacement of [3H]N/OFQ from membranes of HEK cells
expressing recombinant NOP receptor. Details of these assays
have been described previously.16
With respect to MOP receptor efficacy, the rank order for the
methyl substituted derivatives of 1d was 4′ (3c) > 3′ (3b) > 2′
(3a) > H (1d) (Table 2). All three methyl substituted
derivatives were full KOP receptor agonists, whereas NOP
receptor efficacy was in the order 2′ > 3′ = 4′ = H. The 4′-
isopropyl derivative (3d) had low MOP and NOP receptor
efficacy and lower KOP receptor efficacy than the methyl
derivative. The 3′- and 4′-chloro derivatives showed all-round
low efficacy though slightly higher than for the parent. The 3′-
and 4′-fluoro derivatives had low efficacy MOP and NOP
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Table 2. Binding Affinities (Ki, nM) and Maximal Stimulation of [35S]GTPγS Binding of 1d and Analogues to Opioid Receptors
a
b
% stim
KOP
Ki, nM
MOP
6.0
NOP
MOP
KOP
DOP
NOP
1d
3a
3b
3c
3d
3e
3f
1
19
4
3
14
4
4
4
5
6
2
0.17 0.05
0.19 0.08
0.044 0.015
0.16 0.09
43.2 13.4
17
33
50
13
24
14
16
22
18
0
4
90
102
84
34
50
26
17
77
95
30
81
79
79
45
22
19
14
18
9
5
2
3
7
4
4
2
4
1
7
3
6
2
3
1
4
6
1
6
2
0.28 0.16
0.10 0.04
5
3g
3h
3i
12
4
19 10
27
7
2
4a
4b
4c
5
1
5
0.6 0.14
2.8 0.78
1.0 0.22
75 4.2
17
45
3
3
3
44
31
6
6
4
2
3
3
4
3
3
6
1
39 12
6
7
1
−17
0
7
4
0.16 0.04
0.13 0.02
0.39 0.09
0.99 0.43
0.48 0.26
4630 380
1b
20
6
6
26
2
0.089 0.023
212
7
a
Percent maximal stimulation (% stim) at a single high dose (10 μM) with respect to the standard agonists DAMGO (MOP) and U69,593 (KOP)
b
and nociceptin (NOP). Values are an average SEM from three separate experiments. Ki (nM) versus [3H]diprenorphine (for MOP and KOP
receptors) and [3H]N/OFQ (for NOP receptors). Values are an average SEM from three separate experiments.
receptor agonist activity but high efficacy KOP receptor agonist
activity (Table 2).
Screening of the heteroaryl analogues (4, 5, 6, 7) of 1d was
undertaken using the same protocols. The 2-thienyl ligand (4a)
had zero MOP receptor efficacy in the [35S]GTPγS assay,
whereas its 5-methyl (4b) and 5-chloro (4c) substituted
derivatives were MOP receptor partial agonists with efficacy
respectively similar to and significantly higher than that of
buprenorphine (Table 2). 4a had modest KOP receptor
efficacy, whereas 4b and 4c were almost full agonists. 4a had
low NOP receptor efficacy but had binding affinity for this
receptor (Ki = 75 nM) equal to or better than that of
buprenorphine (1b) (Ki = 212 nM). The 3-thienyl analogue
(5) had MOP and NOP receptor efficacy similar to that of 4a
but higher KOP receptor efficacy.
The isomeric 2′- and 4′-pyridyl ligands (6, 7) both had low
efficacy for MOP receptor and NOP receptor, but whereas 7
also had no efficacy for KOP receptor, 6 showed distinct KOP
receptor activity. In binding assays, 7 showed all-round high
affinity for opioid receptors and affinity for NOP receptors
equivalent to 1d.
In Vivo. Compound 1d was evaluated in vivo to confirm the
lack of MOP and KOP receptor agonism. In the hot-plate test
1d showed no antinociceptive activity and instead was an
antagonist of both the MOP receptor agonist morphine and the
Figure 2. Antinociceptive effect using the hot-plate assay in mice of
(A) morphine and (B) EKC in the absence and presence of 10 mg/kg
1d. 1d was given as a 30 min pretreatment. Morphine and EKC were
administered by a cumulative dosing procedure by intraperitoneal (ip)
and subcutaneous (sc) injections, respectively, as described.28 1d was
given ip. Vehicle is a 1:1:9 solution of ethanol, emulphor (oil), and
sterile water.29 Data represent the mean SEM from five to six mice.
KOP receptor agonist ethylketocyclazocine (EKC). At 10 mg/
kg, 1d caused a parallel shift to the right in the dose−response
curve for morphine (Figure 2A), and for EKC there was a
complete flattening of the dose−response curve (Figure 2B).
The effect of 1d antagonism at both receptors was gone by 24
h.
The hot-plate test uses heat as the nociceptive stimulus and
so requires high agonist efficacy in a compound to provide
antinociception.17 Therefore, we checked for agonism in 1d
using the lower agonist efficacy requiring acetic acid stretch test.
In this test 1d also showed no agonist activity (Figure 3A) up
to 32 mg/kg. In contrast buprenorphine was potent and fully
efficacious in this assay (Figure 3B), affording an ED50 value
(determined by nonlinear regression analysis) of 0.16 mg/kg,
similar to the value (0.07 mg/kg) previously reported.18 These
findings confirm that 1d has no, or extremely low, efficacy at
MOP or KOP receptors in vivo.
DISCUSSION
■
In this study of analogues of buprenorphine the aim was to
identify orvinols with zero or very low efficacy for MOP and
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and fluoro (3h, 3i) gave full efficacy agonists. Molecular
modeling of the small-molecule ligands in complex with KOP
receptor in the inactive state (PBD code 4DJH) and in the
activated conformation was performed as described.21 The
molecular models suggest that the ligands such as 1d interact
with the receptor in such a way as to orientate the phenyl ring
into a pocket defined by residues from transmembrane helices
II (Q115, L135), III (C210 and L135), VII (Y312), and
extracellular loops 1 (W124) and 2 (V118, L212) (Figure 4a)
Figure 3. (A) Lack of antinociceptive effect of 1d at 32 mg/kg in the
acetic acid stretch assay in mice. (B) Buprenorphine is a full agonist in
this assay. The assay was performed as described.28 Separate groups of
mice were used for each dose. Data represent the mean SEM from
six mice. Vehicle is as in Figure 2. (∗∗∗) p < 0.001; (∗∗∗∗) p <
0.0001.
KOP receptors together with buprenorphine-like affinity and
efficacy for NOP receptors. The criterion of low MOP and
KOP receptor efficacy was achieved in several orvinols, but the
NOP receptor criterion proved to be very difficult to achieve.
The only ligands with NOP receptor efficacy equal or greater
than that of buprenorphine (1b) had very much higher KOP
receptor efficacy which would be associated with dysphoric side
effects in clinical use. From the little data reported to date,
finding either significant MOP or KOP receptor efficacy in
orvinols that also have affinity and efficacy at NOP receptors is
the norm.19,20 The most interesting candidate is 1d which
satisfies the MOP and KOP receptor efficacy criteria and also
has higher binding affinity for NOP receptors than
buprenorphine. However, its NOP receptor efficacy is lower
than that of buprenorphine. The lack of any activity in the
acetic acid induced abdominal stretch assay, which would be
expected to indicate even low level MOP or KOP receptor
agonist activity and the 52 °C hot-plate antinociceptive assay,
confirms, in vivo, the very low efficacy of 1d at MOP and KOP
receptors and the promise of 1d as a lead for further
investigation.
A surprising finding was the low efficacy of the pyridyl
ligands 6 and 7, in particular the latter, at both KOP and MOP
receptors. It has previously been shown that the 2° alcohols
having opposite relative stereochemistry to buprenorphine, i.e.,
15 (Chart 1), are agonists at both receptors, typically full
agonists at the KOP receptor.13 This view was strengthened
with the finding that 1g, the phenyl ring analogue of 6 and 7,
had high efficacy at KOP receptors as predicted.
SAR at the KOP receptor was striking within this new series.
Efficacy ranged from <20% to 100% with clear differences
between type of substituent and less consistent differences due
to substitution pattern. Of the monosubstituted phenyl
analogues the larger substituents such as i-Pr (3d) and chloro
(3f, 3g) gave the lowest efficacy analogues while methyl (3a 3c)
Figure 4. (a) Predicted binding mode for 1d and analogues (magenta)
in the KOPr in comparison to the crystal structure ligand JDTic
(green). (b) Chloro-substituted analogues of 1d and potential
interactions with the KOPr.
This is the region occupied by the phenolic ring of JDTic in
the reported crystal structure.22 In this antagonist conformation
of the receptor, the presence of substituents on the phenyl ring
appears to be well tolerated, for example, potentially having
interactions with W124 (for 4′-substituents) or Q115 (for 3′-
substituents) or L212 and L135 (for 2′-substituents) (Figure
4b). The KOPr binding pocket in the antagonist conformation,
defined by the binding of JDTic, is large but narrow and deep
and somewhat covered by part of extracellular loop 3.18 It has
been proposed that the active (agonist bound) conformation of
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precipitate that formed was collected by filtration, washed with ether,
and dried under high vacuum.
the receptor provides an even more restricted binding
pocket.21,23 It is therefore possible that the larger substituents,
Cl and i-Pr in this study, cannot readily fit into the agonist
conformation and are therefore predominantly antagonist in
character, whereas the smaller substituents (CH3 and F) are
easily accommodated in the agonist conformation leading to
strong binding to the agonist conformation. However, the low
efficacy of the unsubstituted parent (1d) suggests that a small
substituent is required for good binding to the agonist
conformation.
The C20-ethyl analogues 1c, 1f, 2c, and 2d were all higher
efficacy KOP receptor agonists than their C20-methyl
homologues. Clearly the ethyl group does not provide the
extra bulk around C20 that has been reported to minimize
KOP receptor efficacy13 but is more likely accessing the site
below C8 previously identified as a region associated with KOP
receptor activation.24−26
In vivo evaluation of the lead compound, 1d, confirmed a
lack of agonist activity in the hot-plate test and the acetic acid
stretch assay which is responsive to low efficacy MOP and KOP
receptor agonists. For example, buprenorphine was a fully
effective agonist in this assay. The antagonist action of 1d was
observed as soon as 30 min after administration but dissipated
by 24 h, confirming that 1d is accessing the CNS, in line with
its lower predicted log D7.4 than buprenorphine (4.39 versus
4.81) and its predicted level of brain penetration (ACD/I-lab)
sufficient for CNS activity.
The aim of generating a ligand with a buprenorphine-like
profile but having substantially lower efficacy at MOP receptor
has been achieved in part in the current study. 1d is an
antagonist at MOP and KOP receptors (though it does have
some low efficacy at KOP receptors) and has good affinity,
equivalent or better than buprenorphine, for NOP receptors.
Efficacy at NOP receptors is, however, lower than displayed by
buprenorphine so that the desired profile is not fully realized.
(1′S,5α,6R,7R,14α)-1′-(4,5-Epoxy-7,8-dihydro-3-hydroxy-6-
methoxy-17-cyclopropylmethyl-6,14-ethanomorphinan-7-yl)-
1′-phenylethan-1′-ol (1d). N-CPM dihydronorthevinone 8a (220
mg, 0.52 mmol) in anhydrous toluene (5.2 mL) was treated with
phenylmagnesium bromide (1.5 mL, 1.04 mmol) at room temperature
for 22 h. Purification using column chromatography (30% EtOAc−
petroleum ether−0.5% NH3) gave thevinol 9a (R = Ph), (110 mg,
42%). Rf (30% EtOAc−petroleum ether−0.5% NH3) 0.7. δH (CDCl3)
7.50 (2H, d), 7.33 (2H, t), 7.18−7.26 (1H, m), 6.69 (1H, d), 6.52
(1H, d), 5.50 (1H, s), 4.42 (1H, s), 3.87 (3H, s), 3.61 (3H, s), 2.91
(1H, d), 2.86 (1H, d), 2.39−2.44 (1H, m), 2.11−2.55 (5H, m), 1.87−
1.99 (1H, m), 1.79−1.86 (2H, m), 1.79 (3H, s), 1.54−1.58 (1H, m),
0.77−1.07 (3H, m), 0.55−0.73 (1H, m), 0.33−0.39 (2H, m), −0.10 to
−0.03 (2H, m). δC (CDCl3) 147.46, 146.94, 141.66, 132.76, 128.98,
127.92, 126.79, 126.17, 119.18, 113.97, 97.14, 80.87, 59.54, 57.97,
56.90, 53.00, 48.57, 46.95, 43.52, 36.03, 35.70, 32.65, 30.06, 23.58,
22.72, 17.97, 9.35, 4.18, 3.32. m/z for C32H40NO4, [MH]+ calcd
502.2957. Found 502.2958. 9a (R = Ph) (103 mg, 0.21 mmol) was
treated as in procedure A to yield 1d after silica gel chromatography
(30% EtOAc−petroleum ether−0.5% NH3) (40.0 mg, 39%). Rf (30%
EtOAc−petroleum ether−0.5% NH3) 0.2. δH (CDCl3) 7.50 (2H, d),
7.32 (2H, t), 7.18−7.26 (1H, m). 6.62 (1H, d), 6.45 (1H, d), 5.58
(1H, s), 4.60 (1H, s), 4.42 (1H, s), 3.56 (3H, s), 2.89 (1H, d), 2.84
(1H, d), 2.40−2.42 (1H, m), 2.10−2.19 (5H, m), 1.90−2.08 (1H, m),
1.72−1.84 (3H, m), 1.80 (3H, s), 1.54−1.58 (1H, m), 1.02−1.10 (1H,
m), 0.89−0.94 (1H, dd), 0.69−0.76 (1H, m), 0.56−0.65 (1H, m),
0.30−0.40 (2H, m), −0.1 to 0 (2H, m); δC (CDCl3) 147.27, 132.44,
127.93, 126.83, 126.14, 119.56, 116.51, 97.39, 80.92, 59.52, 58.01,
52.91, 48.48, 47.24, 43.53, 36.10, 35.60, 32.60, 29.95, 23.59, 22.80,
17.97, 9.32, 4.15, 3.31. m/z found [MH]+ 488.2778. C31H38NO4
requires 488.2801. Anal. (C31H38ClNO4) C, H, N.
(1′S,5α,6R,7R,14α)-1′-(4,5-Epoxy-7,8-dihydro-3-hydroxy-6-
methoxy-17-cyclopropylmethyl-6,14-ethenomorphinan-7-yl)-
1′-phenylmethanol (2a). The alcohol 14b (500 mg, 1.03 mmol)
was treated as in procedure A to yield 2a, which was purified by gravity
elution chromatography with MeOH−CH2Cl2. (1:20) (370 mg, 76%).
Rf (MeOH−CH2Cl2, 1:10) 0.48. NMR δH (CDCl3) 0.38−0.40 (2H,
m), 0.40−0.53 (2H, m), 0.64−0.66 (1H, m), 3.01 (1H, d), 3.35 (1H,
d), 3.80 (3H, s), 4.35 (1H, d), 4.65 (1H, d), 5.43 (1H, s), 5.56 (1H,
d), 6.00 (1H, d), 6.43 (1H, d), 6.55 (1H, d), 7.26−7.32 (5H, m). δC
(CDCl3) 3.52, 3.98, 9.18, 23.03, 30.38, 33.00, 42.59, 43.83, 43.92,
47.76, 54.86, 57.04, 59.85, 77.70, 84.53, 97.79, 116.26, 119.83, 124.37,
125.77, 127.70, 128.09, 128.23, 134.32, 137.54, 137.78, 141.71, 146.33.
m/z found M+ for C30H33NO4, 471.2404; calculated 471.2410. Mp
(HCl salt) 227−231 °C (dec, EtOH). Anal. (C30H34ClNO4·H2O) C,
H, N.
(1′R,5α,6R,7R, 14α)-1′-(4,5-Epoxy-7,8-dihydro-3-hydroxy-6-
methoxy-17-cyclopropylmethyl-6,14-ethenomorphinan-7-yl)-
1′-phenylmethanol (2b). The alcohol 12b (550 mg, 1.13 mmol)
was treated as in procedure A to yield 2b which was purified by gravity
elution chromatography with MeOH−CH2Cl2. (1:20) (470 mg, 88%).
Rf (MeOH−CH2Cl2, 1:10) 0.48. NMR δH (CDCl3) 0.01−0.08 (2H,
m), 0.45−0.49 (2H, m), 0.74−0.76 (1H, m), 1.37 (1H, dd), 3.05 (1H,
d), 3.53 (1H, d), 3.69 (3H, s), 4.62 (1H, d), 5.20 (1H, s), 5.49 (1H,
d), 5.81 (1H, d), 6.44 (1H, d), 6.58 (1H, d), 7.31−7.33 (5H, m). δC
(CDCl3) 3.34, 4.20, 9.30, 22.97, 24.99, 33.43, 43.04, 43.41, 44.01,
48.39, 52.24, 56.91, 59.85, 70.18, 80.85, 94.43, 116.41, 119.88, 125.72,
126.44, 126.84, 127.91, 128.18, 134.19, 136.89, 137.41, 143.32, 146.74.
m/z found M+ for C30H33NO4, 471.2408; calculated 471.2410). Mp
(HCl salt) 198−200 °C (dec, EtOH). Anal. (C30H34ClNO4·1.5H2O)
C, H, N, Cl.
(1′R,5α,6R,7R,14α)-1′-(4,5-Epoxy-7,8-dihydro-3-hydroxy-6-
methoxy-17-cyclopropylmethyl-6,14-ethanomorphinan-7-yl)-
1′-(2-pyridyl)ethan-1′-ol (6). 2-Bromopyridine (1.13 mmol) in dry
Et2O was cooled to −78 °C under a nitrogen atmosphere. n-
Butyllithium (1.13 mmol) was added dropwise and the mixture stirred
for 10 min before adding N-CPM dihydronorthevinone (8a, 1 mmol)
in dry THF. The reaction mixture was allowed to warm to room
temperature and stirred for 20 h. After completion, the reaction
EXPERIMENTAL SECTION
■
Reagents and solvents were purchased from Sigma-Aldrich or Alfa
Aesar and used as received. Buprenorphine (1b) was supplied by the
National Institute on Drug Abuse, Bethesda, MD. H and 13C NMR
1
spectra were obtained with a Bruker 400 MHz instrument (1H at 400
MHz, 13C at 100 MHz); δ in ppm, J in Hz with TMS as an internal
standard. Instrumentation was as follows: ESIMS, microTOF
(Bruker); EIMS, Fisons autosampler; microanalysis, PerkinElmer
240C analyzer. Column chromatography was performed using
RediSep prepacked columns with a Teledyne Isco CombiFlash
instrument. Ligands were tested as their hydrochloride salts, prepared
by adding 5 equiv of HCl (1 N solution in diethyl ether) to a solution
of compound in anhydrous methanol. All reactions were carried out
under an inert atmosphere of nitrogen unless otherwise indicated. All
compounds were >95% pure as determined by microanalysis. A
representative synthesis for each series is reported here.
General Procedure A: 3-O-Demethylation with Propane-
thiolate and HCl Salt Formation. A solution of the appropriate
thevinol (0.25 mmol) in anhydrous HMPA (1 mL) under an inert
atmosphere was treated with sodium hydride (21 mg, 0.875 mmol)
followed by 1-propanethiol (79 μL, 0.875 mmol). After the addition
was complete, the reaction mixture was heated to 120 °C and stirred
for 3 h. When the mixture was cooled to room temperature, NH4Cl
(sat., aq) was added and the mixture extracted with diethyl ether. The
organic extracts were washed with water (3×) and brine. The organic
phase was dried (MgSO4), filtered, and evaporated to dryness. The
residue was purified by column chromatography over silica gel. The
HCl salts were prepared by the addition of 2 M HCl in diethyl ether
(1.2 equiv) to a solution of the orvinol in diethyl ether. The white
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Journal of Medicinal Chemistry
Article
mixture was quenched with saturated NH4Cl solution (aqueous) and
extracted with EtOAc. Organic layer was washed with brine, dried
(Na2SO4), and evaporated to yield crude product (10) that was
purified by flash chromatography using MeOH/CH2Cl2 (0.5:99.5)
ACKNOWLEDGMENTS
■
This work was funded by the National Institutes of Health
National Institute on Drug Abuse Grant DA07315 (S.M.H.)
and DA03910 (H.M.)
1
(35%). White solid. H NMR (CDCl3) δ 0.08−0.11 (2H, m), 0.45−
0.52 (2H, m), 0.81−0.86 (2H, m), 1.18−1.22 (2H, m), 1.57−1.73
(4H, m), 2.21−2.41 (8H, m), 2.61−2.70 (2H, m), 2.91 (1H, d, J =
18.24 Hz), 3.02 (1H, d), 3.36 (3H, s), 3.82 (3H, s), 4.36 (1H, s), 5.87
(1H, s), 6.47 (1H, d), 6.62 (1H, d), 7.10−7.12 (1H, m), 7.59−7.63
(2H, m), 8.47 (1H, d). This was 3-O-demethylated using general
procedure A to give 6 as a white solid. 1H NMR (CDCl3) δ 0.08−0.11
(2H, m), 0.40−0.51 (2H, m), 0.80−0.88 (2H, m), 1.20−1.27 (2H, m),
1.62 (1H, d), 1.67 (3H, s), 2.02−2.41 (8H, m), 2.61 (1H, dd), 2.78
(1H, dt), 2.91 (1H, d), 3.02 (1H, d), 3.35 (3H, s), 4.39 (1H, s), 4.55
(1H, bd), 5.83 (1H, s), 6.43 (1H, d), 6.61 (1H, d), 7.10−7.14 (1H,
m), 7.62−7.64 (2H, m), 8.48 (1H, d). 13C NMR, 400 MHz (CDCl3) δ
3.21, 4.33, 9.35, 16.42, 22.45, 28.39, 28.86, 29.92, 35.24, 35.65, 43.81,
46.78, 49.36, 52.28, 57.95, 59.89, 80.1, 97.70, 116.01, 119.37, 120.54,
121.55, 128.54, 132.45, 135.78. 136.93, 145.30, 147.32, 166.46. HRMS,
m/z for (C30H36N2O4), [MH]+: calcd 489.2753, found 489.2821. Anal.
(C30H36Cl2N2O4·3H2O) C, H, N.
ABBREVIATIONS USED
■
MOP, μ opioid; DOP, δ opioid; KOP, κ opioid; NOP,
nociceptin opioid peptide; EKC, ethylketocyclazocine; N/
OFQ, nociceptin/orphanin FQ
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1
chromatography. H NMR δH (CDCl3) 0.01−0.08 (2H, m), 0.45−
0.53 (2H, m), 0.74−0.76 (1H, m), 1.36 (1H, dd), 3.07 (1H, d), 3.53
(1H, d), 3.72 (3H, s), 3.83 (3H, s), 4.62 (1H, d), 5.23 (1H, s), 5.52
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119.28, 125.81, 126.78, 128.16, 128.43, 134.46, 136.85, 141.87, 143.47,
148.30. Found M+ for C31H35NO4, 485.2566; calculated 485.2566
N-Cyclopropylmethylnornepenthol (14b). Ketone 13b27 (800
mg, 1.61 mmol) in THF (10 mL) was added dropwise to a solution of
LiAlH4 (2.5 equiv) in THF (10 mL) at room temperature. The
solution was allowed to stir for 16 h before quenching with a solution
of Rochelle salt (10 mL). Extraction with Et2O yielded 14b (700 mg,
90%) which was purified by recrystallization, Rf (EtOAc−hexane, 1:1,
0.5% NH3) 0.48. NMR δH (CDCl3) 0.02−0.07 (2H, m), 0.37−0.45
(2H, m), 0.64−0.69 (1H, m), 3.03 (1H, d), 3.34 (1H, d), 3.83 (3H, s),
3.84 (3H, s), 4.34 (1H, d), 4.63 (1H, d), 5.37 (1H, s), 5.56 (1H, d),
6.04 (1H, d), 6.48 (1H, d), 6.62 (1H, d), 7.26−7.33 (5H, m). δC
(CDCl3) 3.50, 3.91, 9.24, 22.95, 30.43, 33.17, 42.55, 43.77, 44.13,
47.46, 54.97, 56.82, 57.05, 59.87, 77.70, 84.56, 97.62, 113.83, 119.33,
124.62, 127.58, 128.07, 128.17, 128.35, 134.65, 137.72, 141.88, 141.96,
147.82. m/z found M+ for C31H35NO4, 485.2588; calculated 485.2566.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Full experimental details. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 44-(0)1225-383103. E-mail: s.m.husbands@bath.ac.
uk.
Present Address
⊥V.K.: Centre for Chemical and Pharmaceutical Sciences,
Central University of Punjab, Bathinda, India.
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Notes
The authors declare no competing financial interest.
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